The Notch pathway of signal transduction is important for cell fate decisions. Studies from our laboratory first suggested a role for Notch signaling in thymic development, resulting in a model in which immature thymocytes receiving a Notch signal develop into CD8+ T cells, whereas T cells not receiving a Notch signal mature into CD4+ T cells. Proteolytic processing of the Notch-1 receptor is required for generation of Notch-1-mediated signals, and Delta, a Notch ligand, undergoes proteolytic processing in Drosophila. However, whether processing of mouse Notch homologues and Notch ligands occurs and all the proteases responsible for such processing are not known. The metalloproteases Kuzbanian (Kuz) and TACE have been implicated in processing of Notch receptors and ligands. As processing of Notch is required for Notch signaling, a block in processing of Notch ligands or receptors might result in a decrease in Notch signaling. We will identify the potential Notch receptor/ligand substrates of Kuz and TACE expressed in the thymus and analyze the functional consequences of Notch receptor/ligand cleavage on thymic development. We predict that in thymocytes overexpressing a dominant-negative Kuz or TACE gene, more CD4+ T cells than CD8 T cells might develop. Reciprocally, in thymocytes overexpressing wild-type Kuz or TACE, more CD8+ T cells than CD4+ T cells will develop. These predictions will be tested in vitro and in transgenic mice. Expression and processing of the known Notch receptors and ligands in the thymus will be assessed by quantitative Western blotting, and T cell development assessed by flow cytometry. Correlations between processing and T cell development in these systems will be investigated.